Abstract The overall goals of this research proposal are to define specific pathways of sphingolipid metabolism, define specific roles, define mechanisms, and help place the field of bioactive lipids on solid mechanistic grounds. Based on strong ongoing data, we propose the overall hypothesis that individual enzymes of ceramide metabolism serve as transducers of specific inputs, and the product bioactive sphingolipids function to mediate key responses. This proposal will develop this paradigm in the case of the DNA damage response and other stimuli activating neutral sphingomyelinases in distinct compartments leading to formation of ceramide which acts primarily in a cell cycle checkpoint. We propose the following specific goals: 1. What are the mechanisms involved in induction of nSMase2 and Isc1? 2. How are nSMase2 and Isc1 activated in the nucleus? Is there a specific role for nuclear PIP2? 3. Define mechanisms of regulation and the structure of neutral sphingomyelinases 4. What is the structure of lipids in the nucleus? 5. What is the specific function of nSMase2 (and Isc1) in checkpoints? 6. Do nuclear phosphatases mediate the action of nuclear ceramide; what are the mechanisms involved? 7. Define parallel pathways of bioactive sphingolipids in yeast. Taken together, we endeavor to advance our understanding of bioactive sphingolipids and reduce its complexities to manageable and specific components that promise to shed important light on how these specific pathways function, and their specific roles in stress responses. The ongoing results are defining a totally unexpected role for neutral sphingomyelinases in the eukaryotic DNA damage response through a nuclear lipid-mediated pathway.